After DNA is transcribed and translated into a protein, further post-translational processing involves the attachment of sugar residues, a process known as glycosylation. Different organisms produce different glycosylation enzymes (glycosyltransferases and glycosidases) and have different substrates (nucleotide sugars) available, so that the glycosylation patterns as well as composition of the individual oligosaccharides, even of one and the same protein, will be different depending on the host system in which the particular protein is being expressed. Bacteria typically do not glycosylate proteins and if so only in a very unspecific manner (Moens and Vanderleyden, Arch. Microbiol. 168(3):169-175 (1997)). Lower eukaryotes such as filamentous fungi and yeast add primarily mannose and mannosylphosphate sugars, whereas insect cells such as Sf9 cells glycosylate proteins in yet another way. See R. K. Bretthauer et al., Biotechnology and Applied Biochemistry 1999 30:193-200 (1999); W. Martinet, et al., Biotechnology Letters 1998 20:1171-1177 (1998); S. Weikert, et al., Nature Biotechnology 1999 17: 1116-1121 (1999); M. Malissard, et al., Biochem.Biophys.Res.Comm. 2000 267:169-173 (2000); D. Jarvis, et al., Curr. Op. Biotech. 1998 9:528-533 (1998); and Takeuchi, Trends in Glycoscience and Glycotechnology 1997 9:S29-S35 (1997).
N-linked glycosylation plays a major role in the processing of many cellular and secreted proteins. In eukaryotes, the preassembled oligosaccharide Glc3Man9GlcNAc2 is transferred from dolichol onto the acceptor site of the protein by oligosaccharyltransferase in the endoplasmic reticulum (Dempski and Imperiali, Curr. Opin. Chem. Biol. 6: 844-850 (2002)). Subsequently, the terminal a-1,2-glucose is removed by glucosidase I facilitating the removal of the remaining two a-1,3-glucose residues by glucosidase II (Herscovics, Biochim. Biophys. Acta 1473: 96-107 (1999)). The high mannose glycan remaining is processed by the ER mannosidase, to Man8GlcNAc2, prior to translocation of the glycoprotein to the Golgi, where the glycan structure is further modified. Incorrect processing of the glycan structure in the ER, in turn, can prevent subsequent modification, leading to a disease state. The absence of glucosidase I results in congenital disorder of glycosylation type (CDG) IIb which is extremely rare, with only one reported human case, and leads to early death (Marquardt and Denecke, Eur. J. Pediatr. 162: 359-379 (2003)). Isolation of the Chinese hamster ovary cell line Lec23, deficient in glucosidase I, demonstrated that the predominant glycoform present is Glc3Man9GlcNAc2 (Ray et al., J. Biol. Chem. 266: 22818-22825 (1991)). The initial stages of glycosylation in yeast and mammals are identical with the same glycan structures emerging from the endoplasmic reticulum. However, when these glycans are processed by the Golgi, the resultant structures are drastically different, thus resulting in yeast glycosylation patterns that differ substantially from those found in higher eukaryotes, such as humans and other mammals (R. Bretthauer, et al., Biotechnology and Applied Biochemistry 30:193-200 (1999)). Moreover, the vastly different glycosylation pattern has, in some cases, been shown to increase the immunogenicity of these proteins in humans and reduce their half-life (Takeuchi (1997) supra).
The early steps of human glycosylation can be divided into at least two different phases: (i) lipid-linked Glc3Man9GlcNAc2 oligosaccharides assembled by a sequential set of reactions at the membrane of the endoplasmatic reticulum (ER); and (ii) the transfer of this oligosaccharide from the lipid anchor dolichyl pyrophosphate on to de novo synthesized protein. The site of the specific transfer is defined by an Asparagine residue in the sequence Asn-Xaa-Ser/Thr, where Xaa can be any amino acid except Proline (Y. Gavel et al., Protein Engineering 3:433-442 (1990)).
Further processing by glucosidases and mannosidases occurs in the ER before the nascent glycoprotein is transferred to the early Golgi apparatus, where additional mannose residues are removed by Golgi specific a-1,2-mannosidases. Processing continues as the protein proceeds through the Golgi. In the medial Golgi, a number of modifying enzymes, including N-acetylglucosaminyl-transferases (GnT I, GnT II, GnT III, GnT IV GnT V GnT VI), mannosidase II, and fucosyltransferases, add and remove specific sugar residues. Finally, in the trans-Golgi, galactosyltranferases and sialyltransferases produce a structure that is released from the Golgi. The glycans characterized as bi-, tri- and tetra-antennary structures containing galactose, fucose, N-acetylglucosamine and a high degree of terminal sialic acid give glycoproteins their human characteristics.
When proteins are isolated from humans or animals, a significant number of them are post-translationally modified, with glycosylation being one of the most significant modifications. Several studies have shown that glycosylation plays an important role in determining the (1) immunogenicity, (2) pharmacokinetic properties, (3) trafficking, and (4) efficacy of therapeutic proteins. An estimated 70% of all therapeutic proteins are glycosylated and thus currently rely on a production system (i.e., host) that is able to glycosylate in a manner similar to humans. To date, most glycoproteins are made in a mammalian host system. It is thus not surprising that substantial efforts by the pharmaceutical industry have been directed at developing processes to obtain glycoproteins that are as “humanoid” as possible. This may involve the genetic engineering of such mammalian cells to enhance the degree of sialylation (i.e., terminal addition of sialic acid) of proteins expressed by the cells, which is known to improve pharmacokinetic properties of such proteins. Alternatively, one may improve the degree of sialylation by in vitro addition of such sugars by using known glycosyltransferases and their respective nucleotide sugar substrates (e.g. 2,3 sialyltransferase and CMP-Sialic acid).
Further research may reveal the biological and therapeutic significance of specific glycoforms, thereby rendering the ability to produce such specific glycoforms desirable. To date, efforts have concentrated on making proteins with fairly well characterized glycosylation patterns, and expressing a cDNA encoding such a protein in one of the following higher eukaryotic protein expression systems:    1. Higher eukaryotes such as Chinese hamster ovary cells (CHO), mouse fibroblast cells and mouse myeloma cells (R. Werner, et al., Arzneimittel-Forschung-Drug rResearch 1998 48:870-880 (1998));    2. Transgenic animals such as goats, sheep, mice and others (Dente et al., Genes and Development 2:259-266 (1988); Cole et al., J. Cell. Biochem. 265:supplement 18D (1994); P. McGarvey et al., Biotechnology 13:1484-1487 (1995); Bardor et al., Trends in Plant Science 4:376-380 (1999));    3. Plants (Arabidopsis thaliana, tobacco etc.) (Staub et al., Nature Biotechnology 18:333-338 (2000); McGarvey et al., Biotechnology 13:1484-1487 (1995); Bardor et al., Trends in Plant Science 4:376-380 (1999));    4. Insect cells (Spodoptera frugiperda Sf9, Sf21, Trichoplusia ni, etc. in combination with recombinant baculorviruses such as Autographa californica multiple nuclear polyhedrosis virus which infects lepidopteran cells (Altmann, et al., Glycoconjugate Journal 16:109-123 (1999)).
While most higher eukaryotes carry out glycosylation reactions that are similar to those found in humans, recombinant human proteins expressed in the above mentioned host systems invariably differ from their “natural” human counterpart (Raju, et al. Glycobiology 10:477-486 (2000)). Extensive development work has thus been directed at finding ways to improving the “human character” of proteins made in these expression systems. This includes the optimization of fermentation conditions and the genetic modification of protein expression hosts by introducing genes encoding enzymes involved in the formation of human like glycoforms (Werner et al., Arzneimittel-Forschung-Drug Res. 48:870-880 (1998); Weikert et al. Nature Biotechnology 17:1116-1121 (1999); Andersen et al., Current Opinion in Biotechnology 5:546-549 (1994); Yang et al., Biotechnology and Bioengineering 68:370-380 (2000)).
What has not been solved, however, are the inherent problems associated with all mammalian expression systems. Fermentation processes based on mammalian cell culture (e.g. CHO, Murine, or more recently, human cells) tend to be very slow (fermentation times in excess of one week are not uncommon), often yield low product titers, require expensive nutrients and cofactors (e.g. bovine fetal serum), are limited by programmed cell death (apoptosis), and often do not allow for the expression of particular therapeutically valuable proteins. More importantly, mammalian cells are susceptible to viruses that have the potential to be human pathogens and stringent quality controls are required to assure product safety. This is of particular concern since as many such processes require the addition of complex and temperature sensitive media components that are derived from animals (e.g. bovine calf serum), which may carry agents pathogenic to humans such as bovine spongiform encephalopathy (BSE) prions or viruses.
The production of therapeutic compounds is preferably carried out in a well-controlled sterile environment. An animal farm, no matter how cleanly kept, does not constitute such an environment. Transgenic animals are currently considered for manufacturing high volume therapeutic proteins such as: human serum albumin, tissue plasminogen activator, monoclonal antibodies, hemoglobin, collagen, fibrinogen and others. While transgenic goats and other transgenic animals (mice, sheep, cows, etc.) can be genetically engineered to produce therapeutic proteins at high concentrations in the milk, recovery is burdensome since every batch has to undergo rigorous quality control. A transgenic goat may produce sufficient quantities of a therapeutic protein over the course of a year, however, every batch of milk has to be inspected and checked for contamination by bacteria, fungi, viruses and prions. This requires an extensive quality control and assurance infrastructure to ensure product safety and regulatory compliance. In the case of scrapies and bovine spongiform encephalopathy, testing can take about a year to rule out infection. In the interim, trust in a reliable source of animals substitutes for an actual proof of absence. Whereas cells grown in a fermenter are derived from one well characterized Master Cell Bank (MCB), transgenic technology relies on different animals and thus is inherently non-uniform. Furthermore, external factors such as different food uptake, disease and lack of homogeneity within a herd may affect glycosylation patterns of the final product. It is known in humans, for example, that different dietary habits impact glycosylation patterns, and it is thus prudent to expect a similar effect in animals. Producing the same protein in fewer batch fermentations would be (1) more practical, (2) safer, and (3) cheaper, and thus preferable.
Transgenic plants have emerged as a potential source to obtain proteins of therapeutic value. However, high level expression of proteins in plants suffers from gene silencing, a mechanism by which highly expressed proteins are down regulated in subsequent generations. In addition, it is known that plants add xylose and a-1,3 linked fucose, a glycosylation pattern that is usually not found in human glycoproteins, and has shown to lead to immunogenic side effects in higher mammals. Growing transgenic plants in an open field does not constitute a well-controlled production environment. Recovery of proteins from plants is not a trivial matter and has yet to demonstrate cost competitiveness with the recovery of secreted proteins in a fermenter.
Most currently produced therapeutic glycoproteins are therefore expressed in mammalian cells and much effort has been directed at improving (i.e.g., humanizing) the glycosylation pattern of these recombinant proteins. Changes in medium composition as well as the co-expression of genes encoding enzymes involved in human glycosylation have been successfully employed (see, for example, Weikert et al., Nature Biotechnology 17:1116-1121 (1999)).
While recombinant proteins similar to their human counterparts can be made in mammalian expression systems, it is currently not possible to make proteins with a humanoid glycosylation pattern in lower eukaryotes (e.g., fungi and yeast). Although the core oligosaccharide structure transferred to the protein in the endoplasmic reticulum is basically identical in mammals and lower eukaryotes, substantial differences have been found in the subsequent processing reactions of the Golgi apparatus of fungi and mammals. In fact, even amongst different lower eukaryotes, there exists a great variety of glycosylation structures. This has prevented the use of lower eukaryotes as hosts for the production of recombinant human glycoproteins despite otherwise notable advantages over mammalian expression systems, such as: (1) generally higher product titers, (2) shorter fermentation times, (3) having an alternative for proteins that are poorly expressed in mammalian cells, (4) the ability to grow in a chemically defined protein free medium and thus not requiring complex animal derived media components, and (5) and the absence of retroviral infections of such hosts.
Various methylotrophic yeasts such as Pichia pastoris, Pichia methanolica, and Hansenula polymorpha, have played particularly important roles as eukaryotic expression systems since because they are able to grow to high cell densities and secrete large quantities of recombinant protein. However, as noted above, lower eukaryotes such as yeast do not glycosylate proteins like higher mammals. See, for example, U.S. Pat. No. 5,834,251 to Maras et al. (1994). Maras and Contreras have shown recently that P. pastoris is not inherently able to produce useful quantities (greater than 5%) of GlcNAcTransferase I accepting carbohydrate. (Martinet et al., Biotechnology Letters 20:1171-1177 (1998)). Chiba et al. (J. Biol. Chem. 273: 26298-26304 (1998)) have shown that S. cerevisiae can be engineered to provide structures ranging from Man8GlcNAc2 to Man5GlcNAc2 structures, by eliminating 1,6 mannosyltransferase (OCH1), 1,3 mannosyltransferase (MNN1) and mannosylphosphatetransferase (MNN4) and by targeting the catalytic domain of a-1,2-mannosidase I from Aspergillus saitoi into the ER of S. cerevisiae, by using a ER retrieval/targeting sequence (Chiba 1998, supra). However, this attempt resulted in little or no production of the desired Man5GlcNAc2. The model protein (carboxypeptidase Y) was trimmed to give a mixture consisting of 27% Man5GlcNAc2, 22% Man6GlcNAc2, 22% Man7GlcNAc2, 29% Man8GlcNAc2. As only the Man5GlcNAc2 glycans are susceptible to further enzymatic conversion to human glycoforms, this approach is very inefficient for the following reasons: In proteins having a single N-glycosylation site, at least 73% of all N-glycans will not be available for modification by GlcNAc transferase I. In a protein having two or three N-glycosylation sites, at least 93% or 98%, respectively, would not be accessible for modification by GlcNAc transferase I. Such low efficiencies of conversion are unsatisfactory for the production of therapeutic agents; given the large number of modifying steps each cloned enzyme needs to function at highest possible efficiency.
A number of reasons may explain the inefficiency in the production of glycan formation mentioned above. This may, in part, be due to the inefficient processing of glycans in the ER either by glucosidase I, II or resident ER mannosidase. A recently evolved class of mannosidase proteins has been identified in eukaryotes of the chordate phylum (including mammals, birds, reptiles, amphibians and fish) that is also involved in glucose removal. These glycosidic enzymes have been defined as endomannosidases. The activity of the endomannosidases has been characterized in the processing of N-linked oligosaccharides, namely, in removing a glucose α1,3 mannose dissacharide. The utility in removing of the glucose and mannose residues on oligosaccharides in the initial steps of N-linked oligosaccharide processing is known to be useful for the production of complex carbohydrates has been well-established. Although endomannosidases were originally detected in the trimming of GlcMan9GlcNAc2 to Man8GlcNAc2, they also process other glucosylated structures (FIG. 1). Overall, mono-glucosylated glycans are most efficiently modified although di- and tri-glucosylated glycans may also be processed to a lesser extent (Lubas et al., J. Biol. Chem. 263(8):3990-8 (1988)). Furthermore, not only is GlcMan9GlcNAc2 is the preferred substrate but other monoglucosylated glycans, such as GlcMan7GlcNAc2 and GlcMan5GlcNAc2, are trimmed (to Man6GlcNAc2 and Man4GlcNAc2, respectively) just as efficiently. The occurrence of this class of proteins so late in evolution suggests that this is a unique requirement to enhance the pronounced trimming of N-linked glycans, as observed in higher eukaryotes. This suggestion is further strengthened by the fact that endomannosidase is located in the Golgi and not the ER where complete deglucosylation has traditionally been reported to occur.
Previous research has shown that glucose excision occurs primarily in the ER through sequential action of glucosidase I and II (Moremen et al., Glycobiology 4: 113-125 (1994)). However, more recent research suggests the apparent alternate glucosidase II—independent deglucosylation pathway involving a quality control mechanism in the Golgi apparatus (Zuber et al., Mol. Biol. Cell. Dec;11(12): 4227-40 (2000)). Studies in glucosidase II—deficient mouse lymphoma cells show evidence of the deglucosylation mechanism by the endomannosidase (Moore et al., J. Biol. Chem. 267(12):8443-51 (1992)). Furthermore, a mouse lymphoma cell line, PHAR2.7, has been isolated which has no glucosidase II activity resulting primarily in the production of the glycoforms Glc2Man9GlcNAc2 and Glc2Man8GlcNAc2 (Reitman et al., J. Biol. Chem. 257: 10357-10363 (1982)). Analysis of this latter cell line demonstrated that, despite the absence of glucosidase II, deglucosylated high mannose structures were present, thus, indicating the existence of an alternative processing pathway for glucosylated structures (Moore and Spiro, J. Biol. Chem. 267: 8443-8451 (1992)). The enzyme responsible for this glucosidase-independent pathway has been identified as endomannosidase (E.C. 3.2.1.130). Endomannosidase catalyzes the hydrolysis of mono-, di- and tri-glucosylated high mannose glycoforms, removing the glucose residue(s) present and the juxta-positioned mannose (Hiraizumi et al., J. Biol. Chem. 268: 9927-9935 (1993); Bause and Burbach, Biol. Chem. 377: 639-646 (1996)).
The endomannosidase does not appear to distinguish between differing mannose structures of a glucosylated glycoform, hydrolyzing Glc1Man9-5GlcNAc2 to Man8-4GlcNAc2 (Lubas and Spiro, J. Biol. Chem. 263: 3990-3998 (1988)). To date, the only endomannosidase to have been cloned is from the rat liver. Rat liver endomannosidase encodes a predicted open reading frame (ORF) of 451 amino acids with a molecular mass of 52 kDa (Spiro et al., J. Biol. Chem. 272: 29356-29363 (1997)). This enzyme has a neutral pH optimum and does not appear to have any specific cation requirement (Bause and Burbach 1996, supra). Unlike the glucosidase enzymes, which are localized in the ER, the endomannosidase is primarily localized in the Golgi (Zuber et al., Mol. Biol. Cell 11: 4227-4240 (2000)), suggesting that it may play a quality control role by processing glucosylated glycoforms leaking from the ER.
Given the utility of modifying glucosylated glycans for the production of human-like glycoproteins, a method for modifying glucosylated glycans by expressing an endomannosidase activity in a host cell would be desirable.